This report describes a set of seven informative single-nucleotide polymorphisms (SNPs) and one insertion-deletion (INDEL) distributed over 24 cultivars that can be used for tobacco (Nicotiana tabacum L.) cultivar identification. We analyzed 163,000 genomic DNA sequences downloaded from Tobacco Genome Initiative database and assembled 31,370 contigs and 60,000 singletons. Using relatively long contigs, we designed primer sets for PCR amplification. We amplified 61 loci from 24 cultivars and sequenced the PCR products. We found seven significant SNPs and one INDEL among the sequences and we classified the 24 cultivars into 10 groups. SNP frequency of tobacco, 1/8,380 bp, was very low in comparison with those of other plant species, between 1/46 bp and 1/336 bp. For exact identification of tobacco cultivars, many more SNP markers should be developed. This study is the first attempt to identify tobacco cultivars using SNP markers.